Despite declining fatality rates, atherosclerosis remains the leading killer in industrialized nations. Many attempts have been made to prevent or to reverse atherosclerosis, and also to prevent rapid smooth muscle and other proliferation which may contribute importantly to pulmonary hypertension, transplant rejection, and restenosis after angioplasty or bypass grafting. Such attempts have included the use of antihypertensive and cholesterol-lowering agents, fish oils, corticosteroids, cyclosporin A, heparin and non-anticoagulant heparin fragments, inhibitors of angiotensin-converting enzyme, calcium antagonists, aspirin, prostacyclin and other modulators of platelet and smooth muscle eicosanoid metabolism, colchicine, terbinafine, triazolopyrimidine, analogues of somatostatin, anti-neoplastic agents, seeding with endothelial cells, and devices intended to produce less vascular trauma than balloon angioplasty, such as laser angioplasty and atherectomy. Although preliminary results in experimental animals and in humans show promise for some of these therapies, most will require sustained or local application.
Basic fibroblast growth factor (bFGF) is a protein which has a molecular weight of approximately 16 kD, is acid- and temperature-sensitive and has a high isoelectric point. A structurally related protein, acidic FGF (aFGF), has an acidic isoelectric point. FGFs exhibit a mitogenic effect on a wide variety of mesenchymal, endocrine and neural cells. Of particular interest is their stimulatory effect on collateral vascularization and angiogenesis. Such mitogenic effects have stimulated considerable interest in FGFs as potential therapeutic agents for wound healing, nerve regeneration and cartilage repair, for example.
New capillary growth takes place by a series of sequential steps beginning with the dissolution of the capillary basement membrane. Microvascular endothelial cells stimulated by angiogenic substances, such as BFGF, in vitro secrete collagenase, plasminogen activator, and stromelysin which degrade the basement membrane and allow endothelial cells to migrate toward the angiogenic stimulus. After migrating, the endothelial cells proliferate, develop sprouts, form capillary-like hollow tubules, and finally link tubules into capillary loops.
Many cells that respond to basic FGF have been shown to possess specific receptors on the cell surface membranes. The receptor proteins appear to be single chain polypeptides with molecular weights ranging from 110 to 150 kD, depending on cell type. These receptor proteins bind basic FGF with high affinity (Kd=10-80 pill), and receptor numbers often range from 2000 to 80,000 per cell. Such receptors have been purified from chicken embryo and from rat brain, using a combination of lectin and ligand affinity chromatography and are associated with tyrosine kinase activity, see Imamura et al., B.B.R.C., 583-590 (1989); Huang and Huang, J. Biol. Chem., 261, 9568-9571 (1986).
On baby hamster kidney cells (BHK), two basic FGF receptors with estimated molecular weights of 110 and 130 kD have been reported in Neufeld et al., J. Biol. Chem., 260, 13860-13868 (1985) and Neufeld et al., J. Biol. Chem., 261, 5631-5637 (1986). Although both receptor proteins bind basic FGF and acidic FGF, it appears that the larger receptor protein binds bFGF preferentially and is sometimes referred to as the "high affinity" BFGF receptor; the smaller receptor has somewhat greater affinity for acidic FGF.
The feasibility of using receptor-specific ligands to transport toxins into cells has recently been demonstrated. The strategy, originally applied in immunotherapy by conjugating toxins to monoclonal antibodies (see Blakey et al., Cancer Research, 48, 7072-7078 (1988)), has recently been pursued by coupling toxins with classic endocrine hormones, such as CRF and TRF, with cytokines such as EGF and TGF.alpha. and with lymphokines such as interleukin-2. U.S. Pat. No. 4,468,382 to Bacha et al. shows cytotoxic conjugates wherein the hormone TRH is covalently linked to the toxin CRM 45 by bifunctional cross-linking agents to produce a toxic hybrid protein alleged to be useful in the treatment of certain tumors.
Atherosclerosis, sometimes referred to as arteriosclerosis, results from the development of an intimal lesion and the subsequent narrowing of the vessel lumen. Commonly, atherosclerosis originally appears as a result of the buildup of plaque which lines the interior of blood vessels, particularly the arteries. Whereas bypass surgery is sometimes employed to replace such clogged arteries, in recent years, a number of surgical procedures have been developed so as to interarterially remove such plaque, often by balloon catheterization or other such treatments in which the plaque is either compressed against or scraped away from the interior surface of the artery. Not infrequently, the patient so treated finds a recurrence of such narrowing of the vessel lumen in a relatively short period thereafter, generally referred to as restenosis, requiring a repetition of the surgical procedure to again remove the increasing blockage. A real need exists for preventing such recurrence in patients who have been treated for atherosclerosis.